[ccp4bb] Cif file in coot
Hi All, If I have a pdb with some residues named abc in it, coot can read it and display it well, but I cannot do real space refine to such residues. The tips asks me to import a CIF file, I wonder what is the format of this CIF file? How can I create it and let coot recognise these residues? Or there are some other ways to solve it? Thanks!
Re: [ccp4bb] His tag on membrane protein
In one case, for example, cleaving the N-terminal His-tag improved the solubility of the protein dramatically. Interestingly, expression levels of the protein without the His-tag were much lower than with it! That is also quite common ;-) Look at the second and third codons: optimizing these (as they are optimized in His-tag vectors) can dramatically enhance expression levels. A.
Re: [ccp4bb] Cif file in coot
Dear Yang Li, you probably can download your library file (CIF) from the Hic-up website: http://xray.bmc.uu.se/hicup/ Infact an heterocompound named abc is already existing there... that's a modified acarbose hexasaccharide. Is that the one you were looking for? In this case, here it is: http://ligand-depot.rutgers.edu/pub/ABC/ABC.cif.html Or you can try to let refmac write one for you thanks to a refinement although this one will probably not be an idealised ligand because derived from what's in your structure. But you still will be able to modify it later. Hope this helped, Virgile Quoting yang li [EMAIL PROTECTED]: Hi All, If I have a pdb with some residues named abc in it, coot can read it and display it well, but I cannot do real space refine to such residues. The tips asks me to import a CIF file, I wonder what is the format of this CIF file? How can I create it and let coot recognise these residues? Or there are some other ways to solve it? Thanks! -- ***There are 10 kinds of people: those who can count in binary and the others*** - This mail sent through IMP: http://horde.org/imp/
Re: [ccp4bb] To bathe or not to bathe.
Hi I'm not convinced that the first sentence here has much to do with the second (although both might be true). The main reason was related to absorption. If you didn't completely bathe the crystal in the xray beam, then the diffracting volume of the crystal would be different during the data collection, and thus, scaling would be inaccurate, especially when there was radiation damage. Absorption can be a real problem when the path length through the crystal differs significantly, and is often not closely related to the diffracting volume - think of different paths through a flat plate or a needle. This is the main reason why old-fashioned crystallographers in days of yore used to grind their crystals into a sphere. Absorption problems are exacerbated when you have atoms present in your sample that absorb heavily (this should not be a surprise...). As a former small-molecule crystallographer, I always made sure the crystal was bathed in the beam (so that the diffracting volume was the same), and usually tried to make sure the the whole crystal was in the central part of the beam (to try to make sure I was using the more uniform part of the beam). When I moved to macromolecular work, I found that most people seemed to prefer to get the whole beam going through the crystal, and not worry too much about bits of crystal hanging off outside the beam. There are, of course, reasons for this, among which is that small molecule crystallographers are often spoilt for choice when it comes to picking out the right crystal, and protein crystallographers aren't (at least when I made the switch); they often needed all the crystals they could get in order to get a single dataset - this was in the days before cryo was standard, and room temperature data collection was de rigueur. While practising small molecule crystallography, I still needed to apply absorption corrections to the data, especially where I had loads of strongly absorbing atoms (e.g. in third-row transition metal clusters), in order to both solve and refine the structures. The absorption corrections I used were based on different methods (psi- scans, analytical corrections based on the making precise measurements of the crystal itself, and the (ahem) wonderful Walker Stuart DIFABS (which could turn a pig's ear into a silk purse, in spite of what the purists might say about what it was actually doing to the data!)). just my two ha'porth... This was especially true when you weren't sure that the crystal was well- centered in the xray beam (in a cryostat, and therefore not visible). We typically collected highly redundant data to help compensate for this. We also used to correct for absorption by assigning Bragg indices to the crystal and making precise measurements of crystal dimensions. Scaling programs are now more extensive, and include options to calculate a pseudo-absorption surface. In principle, if you have a beam that is ALWAYS smaller than the crystal, then the same crystal volume is illuminated by the xray beam, and will minimize scaling errors. Bernie Santarsiero On Fri, November 23, 2007 4:34 pm, Jim Pflugrath wrote: It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better.
Re: [ccp4bb] Cif file in coot
Dear Yang Li, What to do depends on whether what you call ABC is the same thing as is in the Refmac dictionary. Refmac thinks ABC is for 'MODIFIED ACARBOSE HEXASACCHARIDE'. If that is what you have - and the atom names match, then simply Get Monomer - ABC. That will run libcheck and refmac to produce a full cif dictionary and read it. Now your all set to tug around your own ABCs. If your ABC is not the same as Refmac, then let Refmac have a bash at your ABC, as Virgile suggest. The CIF description from ligand-depot is pretty useless - less than minimal - and not particularly helpful to Coot or Refmac as it stands. Paul. Virgile Adam wrote: Dear Yang Li, you probably can download your library file (CIF) from the Hic-up website: http://xray.bmc.uu.se/hicup/ Infact an heterocompound named abc is already existing there... that's a modified acarbose hexasaccharide. Is that the one you were looking for? In this case, here it is: http://ligand-depot.rutgers.edu/pub/ABC/ABC.cif.html Or you can try to let refmac write one for you thanks to a refinement although this one will probably not be an idealised ligand because derived from what's in your structure. But you still will be able to modify it later. Hope this helped, Virgile Quoting yang li [EMAIL PROTECTED]: Hi All, If I have a pdb with some residues named abc in it, coot can read it and display it well, but I cannot do real space refine to such residues. The tips asks me to import a CIF file, I wonder what is the format of this CIF file? How can I create it and let coot recognise these residues? Or there are some other ways to solve it? Thanks!
Re: [ccp4bb] To bathe or not to bathe.
One additional point to add not raised by Bob is that crystals are different. So you can shoot at one end of the crystal and say have a mosaicity of 0.2 degrees but somewhere else it might be 1.4 or even worse. In such cases e.g. rod like needles it pays off to have a smaller than crystal beam and walk over you crystal for the best spot to collect your dataset. Jürgen Robert Sweet wrote: Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection and also some other concerns about beam homogeneity in its cross section. How serious is this nowadays ? Can processing programs easily overcome, in a certain mounting, the fact that not all crystal orientations have the same number of unit cells exposed to x-rays ? What about inhomogeneities at the beam ? I understand that technical difficulties may lead you to exposed your crystal partially to the beam, etc..., but how hard should we care about this (how much effort to avoid this) ? The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) = -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
